Process for producing sugar chains using beta1,3-N-acetylglucosaminyltransferase

a technology of acetylglucosaminyltransferase and sugar chain, which is applied in the field of new polypeptides, can solve the problems of difficult to synthesize a large amount of poly-n-acetylglucosamine sugar chains, no reports to date on the efficient production of recombinant glycoproteins to which poly, and achieve the effect of efficiently culture the transforman

Inactive Publication Date: 2007-10-09
HISASHI NARIMATSU +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

[0042]An object of the present invention is to provide synthesis of useful sugar chains; medicaments such as antiinflammatory agents, anti-infective agents and tumor metastasis inhibitory agents; foods such as dairy products; a method for improving protein stability, and the like; and a method for diagnosing inflammatory diseases and types and malignancies of cancers, by use of a novel polypeptide having a β1,3-N-acetylglucosaminyltransferase activity.

Problems solved by technology

Also, it has not been found whether β3GnT uses lactosylceramide and paragloboside as its substrates inside cells.
Although a partially purified β1,3-N-acetylglucosaminyltransferase has been used for the synthesis of the poly-N-acetyllactosamine sugar chain moiety of the oligosaccharides, supply of this enzyme is a limiting factor so that it is difficult to synthesize a large amount of poly-N-acetyllactosamine sugar chains [Glycobiology, 7, 453 (1997)].
However, there are no reports to date on the efficient production of recombinant glycoproteins to which poly-N-acetyllactosamine sugar chains are added, in host cells suitable for the production of recombinant glycoproteins (e.g., Namalwa cell, Namalwa KJM-1 cell, CHO cell).

Method used

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  • Process for producing sugar chains using beta1,3-N-acetylglucosaminyltransferase
  • Process for producing sugar chains using beta1,3-N-acetylglucosaminyltransferase
  • Process for producing sugar chains using beta1,3-N-acetylglucosaminyltransferase

Examples

Experimental program
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Effect test

example 1

Cloning of β1,3-galactosyltransferase Homologue (Rat G6) Gene from a Rat Tibia-derived cDNA Library

(1) Preparation of RNA

[0432]From rat tibia, 2.2 mg of total RNA was prepared by the guanidine thiocyanate-cesium trifluoroacetate method [Methods in Enzymology, 154, 3 (1987)]. Next, 15.7 μg of mRNA was obtained as poly(A)+ RNA by passing 2.0 mg of the total RNA through an oligo(dT) cellulose column (manufactured by Collaborative Research).

(2) Preparation of cDNA Library

[0433]Using 4.0 μg of the mRNA obtained in the above (1), synthesis of cDNA, ligation of BamHI adapter and digestion with NotI were carried out according to the linker primer method [Preparation Methods of Gene Library (Idenshi Library no Sakusei-ho), edited by Hirosho Nojima, Yodo-sha, 1994]. A cDNA library in which 5′-terminal of cDNA is always present in the BamHI site side of the vector was constructed by inserting the thus obtained double-stranded cDNA between BamHI site and NotI site of a plasmid pBluescript II SK...

example 2

Search of Gene Encoding Human G6

[0436]Human G6 gene corresponding to the rat G6 cDNA obtained in Example 1 was searched. As a result of the search of genes having homology with the nucleotide sequence (represented by SEQ ID NO:3) of the cDNA contained in OVX2-038 obtained in Example 1, or genes having a possibility to encode polypeptides having homology with the polypeptide (represented by SEQ ID NO:32) considered to be encoded by the cDNA at amino acid level from gene data bases by using the programs of BLAST [J. Mol. Biol., 215, 403-410 (1990)] and FrameSearch [manufactured by Compugen], one EST (expressed sequence tag) sequence (GenBank No. AI039637) was found. Since the EST sequence is 428 bp, a total amino acid sequence of a polypeptide encoded by a gene corresponding to this EST and the function of the polypeptide cannot be found from this sequence information alone.

example 3

Cloning of Human G6 cDNA

[0437]Cloning of candidate gene fragments was attempted by designing a primer set specific for the EST sequence (GenBank No. AI03937) found in Example 2. As the primers, CB-462 having the nucleotide sequence represented by SEQ ID NO:4 and CB-464 having the nucleotide sequence represented by SEQ ID NO:5 were used. Using the primer set, the presence of human G6 cDNA was examined by PCR by using single-stranded cDNAs prepared from various organs, or various cDNA libraries, as templates. As a result, a DNA fragment of about 250 bp was amplified when a cDNA library derived from a human colon cancer cell line Colo205 or a cDNA library derived from human gastric mucosa was used as a template.

[0438]A human G6 cDNA clone of about 3 kb was obtained by screening the above two cDNA libraries by using the amplified fragment as a probe. Since the clone was lacking the 5′-terminal portion, a 5′-terminal side DNA of the human G6 cDNA was obtained by using 5′ RACE method. Ful...

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Abstract

The present invention provides a novel polypeptide having a β1,3-N-acetylglucosaminyltransferase activity, an agent for synthesizing a sugar chain comprising the polypeptide, a process for producing a sugar chain or a complex carbohydrate using the agent for synthesizing a sugar chain, DNA encoding the polypeptide, a process for producing the polypeptide, an antibody against the polypeptide, and a diagnosis method and a medicament for treatment for inflammation, cancer or tumor metastasis using the DNA or the antibody. The present invention is useful for synthesis of a useful sugar chain and diagnosis and treatment for inflammatory diseases, cancer or tumor metastasis.

Description

TECHNICAL FIELD[0001]The present invention relates to a novel polypeptide having a lactosylceramide β1,3-N-acetylglucosaminyltransferase activity and a paragloboside β1,3-N-acetylglucosaminyltransferase activity; an agent for synthesizing a sugar chain, which comprises the polypeptide as an active ingredient; a DNA encoding the polypeptide, an agent for detecting inflammation, cancer or tumor metastasis, which comprises the DNA; a recombinant DNA obtainable by inserting the DNA into a vector; a transformant comprising the recombinant DNA; a process for producing the polypeptide using the transformant; a process for producing a sugar chain or complex carbohydrate using the polypeptide; a process for producing a sugar chain or complex carbohydrate using the transformant; a method for detecting inflammation, cancer or tumor metastasis using an oligonucleotide obtainable from a DNA encoding the polypeptide; an antibody which recognizes the polypeptide; a method for immunohistostaining u...

Claims

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Application Information

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Patent Type & Authority Patents(United States)
IPC IPC(8): C12P19/00C07H21/04C12N1/20C12N15/00C12N9/10C12N1/11C12N9/12C12N15/54
CPCC12Y204/0108C12N9/1241C12N9/1051A01K2217/05A01K2217/075C07K2319/00C12N2799/026
Inventor NARIMATSU, HISASHISASAKI, KATSUTOSHINATSUME, AYUMIMIO, HIROYUKINAKAGAWA, SATOSHISEKINE, SUSUMUTOGAYACHI, AKIRA
Owner HISASHI NARIMATSU
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